Project Summary/Abstract The ultimate goal of this proposal is to develop an innovative and modular technology for osteochondral tissue repair comprising injectable, thermally responsive, in situ forming, and biodegradable hydrogel constructs capable of sustaining the delivery of encapsulated chondrogenic and osteogenic cell populations in a spatially directed fashion to promote native tissue regeneration. We hypothesize that a cytocompatible hydrogel system consisting of non-shrinking, injectable hydrogels with fully soluble degradation products will be formed through the combination of custom poly(N-isopropylacrylamide)-based thermogelling macromers and lysine-based crosslinking macromers that also contain sites for covalent attachment of chondroitin sulfate (CS) to enhance the integration of resultant constructs. Additionally, we hypothesize that the incorporation of poly(L-lysine) (PLL) within the thermogelling hydrogel will enhance the chondrogenic capacity of co-encapsulated articular chondrocyte and mesenchymal stem cell (AC-MSC) cocultures via the induction of developmentally relevant condensation signals. Finally, we hypothesize that a bilayered construct combining the CS-modified chondrogenic hydrogel layer with an osteogenic hydrogel layer of designer mineralizing capability will be leveraged to promote effective osteochondral tissue repair. Three Specific Aims are proposed to address these hypotheses. First, a lysine-based polyesterurethane macromer comprising a biodegradable poly(DL-lactic-co- glycolic acid) mid-block and chemically crosslinkable diamine functionalities will be developed, covalently modified with CS, combined with the thermogelling macromer and thoroughly assessed to establish structure- property relationships. Second, PLL will be incorporated into the hydrogels and its effects on the chondrogenesis of encapsulated AC-MSC cocultures will be evaluated. Further, the combined effects of PLL presentation, AC-MSC coculture, and CS-modification of hydrogel constructs on cartilage tissue integration will be also evaluated ex vivo. Third, the hydrogels developed in Specific Aim 1 and optimized for chondrogenic potential in Specific Aim 2 will be merged with hydrogel formulations with high mineralizing capability to yield bilayered hydrogel constructs comprising chondrogenic and osteogenic layers for the effective repair of osteochondral defects. The potential synergistic effects of encapsulated cells in the osteogenic and chondrogenic layers with PLL delivery will be evaluated in vitro and in vivo to determine the most effective configuration for osteochondral tissue repair in a well-established rabbit osteochondral defect model. The proposed system will address persisting significant challenges associated with osteochondral defect repair by enabling stable integration of the construct with the surrounding native cartilage tissue through a highly modular two-component design, while promoting the chondrogenic and osteogenic differentiation of respective cell populations delivered to effect both cartilage and bone regeneration, respectively.